Studies on the functional basis of topoisomerase 1 and BRCA1 association in the response to CPT
Tohme, Yara Hamade
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Topoisomerase I inhibitors such as camptothecins (CPTs) are a potent anti-neoplastic drug and are currently being used in clinics. However, the efficiency of this drug remains low, only 13-30% of patients respond to the drug. It has been shown that topol degradation by ubiquitin proteasomes pathway provides drug resistance. Our lab has shown that breast cancer susceptibility gene BRCA 1 ubiquitinates topol and initiates the degradation of topol in response to the drug. Topol ubiquitination by BRCA 1 is understood however the precise mechanism is not known. To understand the mechanism of resistance of this drug the present study aims to characterize the association between topoisomerase I (topol) and BRCA1 in the response to Camptothecin (CPT). A cell viability assay was performed to demonstrate the relationship between rate of topol degradation and response to CPT. GST-pull down and Immunoprecipitation experiments were performed to determine the nature of topol and BRCA1 association. Co-localization experiments were performed to observe the interaction of the proteins in the cell in response to CPT. It was observed that the rate of topol degradation determined the response to CPT. In the response to CPT, topol and BRCA 1 form a protein complex and BRCA 1 acts as an E3-ligase to ubiquitinate topol which is degraded by the ubiquitin proteasome pathway (UPP). Also, it was observed that BRCA1 and topol colocalize in the cell nucleus in the CPT response. The association of topol and BRCA 1 has functional implications in the degradation of topol and understanding this association could lead to the development of new drugs that could prevent topol degradation thereby enhancing CPT efficiency.
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